School of Biomedical Sciences, The University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom.

Abstract

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.

Within a peptide backbone, non-enzymatic deamidation of an asparagine-Xaa linkage or dehydration of an aspartic acid-Xaa linkage can give rise to a five-carbon succinimide (indicated by the dashed arrows). Succinimide hydrolysis (OH−) yields an isoaspartate residue (major product, indicated by the dashed arrow), or an aspartic acid (minor product, indicated by the right solid arrow). PIMT utilises SAM to carboxyl-methylate the isoaspartate forming a methyl ester that readily hydrolyses under physiological conditions to liberate methanol and reform the five-carbon succinimide (lower solid arrow). Subsequent succinimide hydrolysis will again yield isoaspartate or ‘repaired’ aspartate products (right solid arrow). PIMT activity is influenced by the levels of SAM and SAH, which are likewise governed by the enzyme and metabolite levels of the methionine metabolic pathway.

Identical amino acids are marked with an asterisk. The positions of the human α-synuclein familial mutants A30P and A53T are underlined. Aspartic acid and asparagine residues that may give rise to isoaspartate formation are shown in bold.

Five µg of 20 day in vitro aged α-synuclein (upper panels) or 5 µg of 20 day in vitro aged β-synuclein (lower panels) were methylated with 3H-SAM using exogenous PIMT, and proteins resolved by 2D-PAGE. Proteins were stained with Coomassie (left panels), and methylation of isoaspartate visualised by autoradiography (right panels).

Quantitation of isoaspartate protein damage after in vitro ageing of synucleins.

Native α-synuclein, β-synuclein, A30P mutant α-synuclein, or A53T mutant α-synuclein were in vitro aged over a time course of 20 days. Two and a half µg of protein was removed after 0, 2, 5, 9, and 20 days of in vitro ageing and the level of isoaspartate formation quantified by a methanol diffusion assay.

Quantitation of isoaspartate formation during in vitro ageing of α-synuclein either with or without co-incubation with β-synuclein, and co-incubation autoradiography.

Alpha-synuclein, A30P mutant α-synuclein, or A53T mutant α-synuclein were in vitro aged either with or without β-synuclein over a time course of up to 20 days. Two and a half µg of protein was removed after 0, 2, 5, 9, and 20 days of in vitro ageing and the level of isoaspartate formation quantified by a methanol diffusion assay (left panels). Black filled columns correspond to α-synuclein isoaspartate values, grey filled columns correspond to isoaspartate levels from α-synuclein co-incubated with β-synuclein. Co-incubation of β-synuclein with α-synucleins significantly reduced total isoaspartate formed during the in vitro ageing time course. Significance is marked with an asterisk: *p<0.01; **p<0.001. Synuclein proteins from co-incubation experiments were methylated with 3H-SAM using exogenous PIMT, resolved by 1D PAGE, and isoaspartate methylation visualised by autoradiography (right panels). Stable α-synuclein protein oligomers that contained isoaspartate peptide linkages are marked with arrowheads.

(A) Alpha, β, or co-incubated mixtures of both proteins were resolved by size exclusion chromatography on a Superose 12 column. Representative chromatograms from a buffer blank, and 20 day in vitro aged α-synuclein, β-synuclein, and co-incubation mixtures are shown. Synuclein proteins eluted as a single peak (Peak 1) in two column fractions at an elution volume of ∼10.89 ml, which corresponds to a molecular weight of ∼57.5 kDa. Peak 2 eluted at ∼15.20 ml, and was an artefact of the in vitro ageing buffer. The migration of proteins of known molecular weight was used to calibrate the column – see inset. (B) Peak column fractions were concentrated, resolved by 1D-PAGE, and stained with colloidal Coomassie blue (upper section), and Western blotted using specific anti-synuclein antibodies (middle section). Beta-synuclein was not immunoprecipitated from either column fractions or aged synuclein reactions when control serum was applied, and β-synuclein antibody did not cross-react with α-synuclein protein. Half of a µg of β-synuclein was similarly blotted to provide a protein standard (lower section, left panel). Beta-synuclein was immunoprecipitated directly from in vitro aged fractions, with the level of β-synuclein recovered confirmed by Western blotting. Parent β-synuclein protein as well as lower molecular weight proteolytic fragments were recovered (marked with arrowheads). Two and a half µg of β-synuclein was similarly blotted to provide a protein standard (lower section, central panel). Beta-synuclein immunoprecipitated from in vitro aged co-incubated mixtures, or from Superose 12 column peak fractions also detected the presence of α-synuclein by Western blotting. Two and half µg of α-synuclein was similarly blotted to provide a protein standard (lower section, right panel). The positions of protein molecular weight standards are also included on Western blots.